Int.J.Curr.Microbiol.App.

Sci (2017) 6(7): 2295-2306

International Journal of Current Microbiology and Applied Sciences

ISSN: 2319-7706 Volume 6 Number 7 (2017) pp. 2295-2306
Journal homepage: http://www.ijcmas.com

Original Research Article https://doi.org/10.20546/ijcmas.2017.607.330

Pathological and Ultra-structural Changes in Testis of Rats due to

Doxorubicin Toxicity and its Amelioration with Quercetin
S. Divya1, D. Madhuri1*, M. Lakshman1 and A. Gopal Reddy2
1
Department of Veterinary Pathology, College of Veterinary Science, Rajendranagar,
Hyderabad-500030, Telangana, India
2
Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science,
Rajendranagar, Hyderabad-500030, Telangana, India
*Corresponding author

ABSTRACT

The protective effect of quercetin on doxorubicin induced testicular tissue damage was
Keywords investigated using histopathological and ultra structural pathology approaches. A total of
48 male albino rats (Wistarstrain) were randomly divided into 4 groups consisting of 12 in
Rats, Testis,
Doxorubicin, each group. Group 1-Control, Group 2-Doxorubicin treated @4mg/kg b.wt
Quercetin, intraperitoneally weekly once. Group 3-treated with quercetin @ 80mg/kgb.wt, orally
Histopathology daily. Group 4-treated with doxorubicin @ 4 mg/kg b.wt, intraperitoneally weekly once
and ultrastructural and quercetin @ 80mg/kg b.wt, orally daily. The histopathological studies of group 2
pathology. showed loss of germ cells with vacuolation in semniferous tubules, coagulative type of
necrosis and edema in the interstitial spaces. In later stages severe atrophy of semniferous
Article Info tubules with loss of germ cells and giant cells were noticed. In group 4 spermatogenesis
Accepted: with several mitotic figures along with mild degenerative changes were noticed. The ultra-
26 June 2017 structural-studies of group 2 revealed margination of chromatin, swelling of mitochondria
Available Online: and vacuolar changes in cell cytoplasm and on 29 th severity was increased with complete
10 July 2017 loss of cell integrity. In group 4 improvement was noticed with appearance of germ cells
and sperm cells.

Introduction
Anthracyclins are currently the most effective
group of antineoplastic drugs used in clinical
practice, among them Doxorubicin (DOX), is
a key chemotherapeutic agent in cancer
treatment, isolated from the soil fungus
Streptomyces peucetiuscaesius. Doxorubicin,
known as topoisomerase II (TOP2) poison,
blocks the synthesis of DNA by intercalating
into the DNA strand. Although doxorubicin is
considered a very potent and efficient
chemotherapeutic drug, it also kills healthy
cells, especially those under rapid and
constant proliferation. Therefore, DNA of
rapidly dividing cells such as the testicular
germ cells can be the preferential target of
doxorubicin resulting in reproductive toxicity.
DOX-induced organopathy involves the
generation of free radicals which result in
membrane and macromolecule damage by
lipid peroxidation, DNA fragmentation and
protein oxidation (Granados-Principal et al.,
2010). Because of high concentration of
polyunsaturated fatty acids and low
antioxidant capacity the mammalian

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 Int.J.Curr.Microbiol.App.Sci (2017) 6(7): 2295-2306
spermatozoa are more vulnerable to oxidative
damage (Vernet et al., 2004). Dox causes an
imbalance between free oxygen radicals
(ROS) and antioxidants enzymes resulting in
tissue injury.
Quercetin (3, 3´, 4´, 5, 7- pentahydroxy-
flavones) is a plant pigment an important
dietary flavonoid found in a variety of plant-
based foods such as Red-onions, broccoli,
apples, cherries, berries and tea. It is
considered to be a strong antioxidant due to
its ability to scavenge free radicals and bind
transition metal ions (Hollman and Katan
1997; Sakanashi et al., 2008).
Hence the objective of this study was to
assess the reproductive toxicity in male albino
Wistar rats induced by doxorubicin and
ameliorative effect of quercetin to overcome
the doxorubicin induced testicular toxicity
Materials and Methods
A total of 48 male albino rats (Wistarstrain)
weighing 250-280g were procured from
Sanzyme laboratories Ltd., Hyderabad and
were randomly divided into 4 groups
consisting of 12 in each group. Group 1-
Control, Group 2-Doxorubicin treated @
4mg/kg b.wt intraperitoneally weekly once.
Group 3-treated with quercetin @ 80mg/kg
b.wt, orally daily. Group 4-treated with
doxorubicin @ 4 mg/kg b.wt,
intraperitoneally weekly once and quercetin
@ 80mg/kg b.wt, orally daily. The
experiment was carried out according to the
guidelines and prior approval of the
Institutional Animal Ethics Committee
(IAEC).
The animals were sacrificed at fortnight
intervals. From each group, 6 rats were
sacrificed on 15th day and remaining were
sacrificed on 29thday. The abdominal cavity
was opened through a midline abdominal
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incision to expose the reproductive organs.
Then the testes were excised and trimmed of
all fat. The gross pathology and testes weights
of each animal were evaluated. The testes
from all the groups were collected for
histopathological and ultra-structural studies
in suitable preservatives. For
histopathological examination the testes
samples were collected and fixed in 10%
neutral buffer formalin (NBF) soon after
sacrifice. The samples were processed,
sectioned (5μm) and stained with
Hematoxylin and Eosin (H&E) as per the
standard procedure (Luna, 1968). To study
the ultra-structural Pathology the testes
samples were collected and preserved in 2.5%
gluteraldehyde (PBS based EM grade) and
processed for transmission electron
microscopic (TEM) study asper the standard
protocol (Bozzala and Russels, 1998).
Results and Discussion
Absolute testicular weights (g)
Testes weight in different groups on day 15
and 29 is shown in the table 1. Testes weights
(g) were taken immediately after sacrifice and
the weight was significantly (P<0.05) reduced
in doxorubicin treated group (2.09) when
compared with Group 1 (2.88), Group 3
(2.64) and Group 4 (2.43) on 15th day.
Similarly on 29th day also a significant
reduction in testicular weights was noticed in
Group 2 (1.07) compared with control (2.71),
Group 3 (2.60) and Group 4 (1.38). The
testicular weights of Group 1 and Group 3
were comparable with each other.
Relative testicular weights (g)
The mean values of relative testicular weight
was significantly (P<0.05) reduced in Group 2
(0.95, 0.36) compared with Group 1 (1.11,
1.09), Group 3 (1.08, 1.00) and Group 4
(1.10, 0.64) on 15th and 29th day
 Int.J.Curr.Microbiol.App.Sci (2017) 6(7): 2295-2306
respectively. There was no significant
difference between Group 1 and Group 3 on
15th and 29th day of experiment.
Gross pathology
On 15th and 29th day of experiment the
testicular size of Group 2 decreased compared
with Group 1, Group 3 and Group 4.
Histopathology
The examination of testicular sections of
control group on day 15 and day 29 of the
experiment showed normal seminiferous
tubules with active spermatogenesis. Each
tubule is bounded by a basal lamina,
spermatogenic cells and sertoli cells.
Spermatogenic cells were arranged in layers
occupying space between basement
membrane and lumen of tubule (Fig. 1). In
between the tubules i.e. in interstitial space
fibroblasts, collagen, blood vessel and Leydig
cells are present. The histopathological
sections of Group 3 testes showed normal
seminiferous tubules with regular outlines as
noticed in control. They were lined in 4-6
layers of germinal epithelium at different
stages of spermatogenesis (Fig. 2) and the
lining epithelium consisted of sertoli cells. On
day 15 the testicular sections of Group 2
revealed disorganized, disrupted epithelium of
affected tubules and shedding of germ cells in
to the lumen. In most of the seminiferous
tubules vacuolation was observed (Fig. 3). In
some seminiferous tubules complete loss of
cells and necrosis of germ cells was
characterized by presence of large pale
eosinophilic mass and few retained
spermatids were noticed at the base of
seminiferous tubules. In addition to changes
in seminiferous tubules, the interstitial tissue
was widened, edema and vacuoles were
noticed. On day 29 the testicular sections of
Group 2 showed disrupted spermatogenic
cells in many seminiferous tubules. Most of
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the seminiferous tubules were devoid of germ
cells with dilated lumen (Fig. 4). In few
seminiferous tubules syncytia/giant cell
formation was noticed. Almost all the
seminiferous tubules showed severe atrophy
as they were devoid of epithelium (Fig. 5).
In Group 4 the testicular sections on day 15
revealed nearly normal seminiferous tubules
except for widening of interstitial space (Fig.
6). In Group 4 most of the seminiferous
tubules exhibited spermatogenesis (Fig. 7)
and several mitotic figures could be seen in
spermatogenic cells. Most of the seminiferous
tubule showed organized epithelium except in
few tubules mild degenerative changes and
disorganized arrangement of germ cells were
noticed.
Ultra Structure
The ultrastructure examination of rat testis
from control group and Group 3 showed
normal seminiferous tubular structures
surrounded by a thick basal lamina and myoid
cells. The spermatogonia resting on basal
lamina with large nucleus and centrally
placed prominent electron dense nucleolus,
the spermatocytes with spherical nucleus
containing electron dense hetero and
euchromatin masses distributed in the
nucleoplasma and cytoplasm showed
numerous light and dense mitochondria (Fig.
8). The ultrathin sections revealed the
presence of numerous sperms at different
stages with different sizes and shapes. The
Group 2 sections on 15th day revealed
variable degrees of degenerative changes in
germ cells including thin basement
membrane, numerous distorted spermatogenic
cells, cytoplasmic vacuolation with swollen
mitochondria. Few primary spermatocytes
showed mild margination of chromatin
material and presence of abnormal sperms
were appears to be typical (Fig. 9).
 Int.J.Curr.Microbiol.App.Sci (2017) 6(7): 2295-2306

Table.1 Absolute and relative testis weight (g) in different groups on day 15 and
29 of the experimental period

Group Day Absolute Relative testis

testis weight weight
I 15 a a
2.88 ± 0.08 1.11 ± 0.03
II 15 c b
2.09 ± 0.12 0.95 ± 0.05
III 15 ab a
2.64 ± 0.10 1.08 ± 0.03
IV 15 b a
2.43 ± 0.12 1.10 ± 0.05
I 29 a a
2.71 ± 0.03 1.09 ± 0.02
II 29 c c
1.07 ± 0.13 0.36 ± 0.04
III 29 a a
2.60 ± 0.04 1.00 ± 0.02
IV 29 b b
1.38 ± 0.13 0.64 ± 0.05
Values are Mean + SE (n = 6) One way ANOVA
 Means with different superscripts differ significantly (P<0.05)

Fig.1 Group 1 showing the spermatogenic cells arranged in layers occupying space between
Basement membrane and lumen of tubule. H&E 400

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Fig.2 Microphotograph of Group 3 showing normal seminiferous epithelium and interstitial

Tissue with active spermatogenesis and spermatozoa filled in lumen. H&E×400

Fig.3 Microphotograph of Group 2 showing tubular vacuolation with loss of germ cells on
15th day. H&E×400

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Fig.4 Microphotograph of Group 2 showing widening of interstitial space, loss of germinal

Epithelium with dilated lumen of seminiferous tubule on 29th day. H&E×400

Fig.5 Microphotograph of Group 2 showing seminiferous tubules with severe atrophy and
Devoid of germinal epithelium on 29th day. H&E×100

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 Int.J.Curr.Microbiol.App.Sci (2017) 6(7): 2295-2306

Fig.6 Microphotograph of Group 4- showing widening of interstitial space and seminiferous

Tubules were nearly normal with spermatogenesis on 15th day. H&E×100

Fig.7 Microphotograph of Group 4 showing seminiferous tubules exhibiting spermatogenesis on

29th day. H&E×100

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 Int.J.Curr.Microbiol.App.Sci (2017) 6(7): 2295-2306

Fig.8 TEM of Group 1 showing spermatogonia (sg) resting on basement membrane (bm),
Primary spermatocytes (ps) and round spermatids (rs).UA&LC2895x

Fig.9 TEM Group 2 showing mild margination of chromatin (arrow) and numerous abnormal
Sperms (abs).UA & LC 2895x

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 Int.J.Curr.Microbiol.App.Sci (2017) 6(7): 2295-2306

Fig.10 TEM Group 2 on 29thday showing loss of germ cells and severe swelling of
Mitochondria (m).UA & LC 3860x

Fig.11 TEM Group 4 showing numerous sperm cells (s) of different size and shapes.
UA & LC 2895x

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 Int.J.Curr.Microbiol.App.Sci (2017) 6(7): 2295-2306
Uniform electron dense lipid bodies were also
observed within germ cells. Severe cellular
changes like indistinct cellular junctions,
absence of spermatocytes and spermatids
were observed on 29th day (Fig. 10) in
addition to the changes noticed on 15th day in
Group 2. The ultrathin sections of testis in
Group 4 on 15th day revealed the presence of
spermatogenic cells which appeared to be
near normal except with swollen
mitochondria. Some spermatogenic cells have
showed initiation of acrosomal cap like
structure in addition to increase in number of
sperm cells (Fig. 11). On 29th day
spermatogenesis was evident with presence of
spermatogonia, spermatocytes, spermatids
and sperm cells. Few germ cells showed mild
to moderate degenerative changes like
vacuolated cytoplasm, swollen mitochondria
and electron dense bodies masked over
nucleus.
The present study revealed DOX induced
testicular damage as in Group 2 there was
significant reduction in absolute and relative
testicular weights compared to control. The
observations in Group 2 were in accordance
with the earlier reports of Kato et al., (2001),
Atessahin et al., (2006), Saalu et al., (2009)
and Badkoobeh et al., (2013). It is thought
that severe parenchymal atrophy in the
seminiferous tubules, reduced number of
germ cells, atrophy of Leydig cells and lower
rate of spermatogenesis after doxorubicin
administration in rats causes reduction in
testicular weights. The testicular weights of
Group 4 were significantly higher than Group
2 indicating protective action of quercetin.
Similar findings were reported by Altintas et
al., (2015) where quercetin is used as
ameliorative agent against docetaxel. Grossly
the testicular size was markedly decreased in
Group 2 compared with Group 1, which could
be due to severe toxic action of DOX on
seminiferous tubules. Similar findings were
reported by Yeh et al., (2007). Testicular
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histopathology showed a detectable effect of
DOX on spermatogenesis. On day 15 the
testicular sections revealed disorganized,
disrupted germinal epithelium and shedding
of germ cells in to the lumen of tubules. This
could be attributed to DOX induced oxidative
stress and production of free radicals, this
agrees with findings reported by earlier
workes (Bashandy and Amin, 2012). In most
of the seminiferous tubules vacuolation and
severe coagulative necrosis with loss of
spermatogenic cells was observed, indicating
the degeneration of germcells.
These findings are in accordance with the
earlier scientists (kato et al., (2001), Patil et
al., (2009), Bashandy and Amin (2012), and
Ahmed et al., (2013). The interstitial tissue
was widened due to severe edema and
vacuoles were noticed, this could be attributed
to endothelial dysfunction and oxidative stress
in the vascular wall as reported by
Venderamani et al., (2010) and Brilhantae et
al., (2011). On day 29 the testicular sections
showed disrupted spermatogenic cells in
many seminiferous tubules. Most of the
seminiferous tubules were devoid of germ
cells with dilated lumen, indicated that
reserve and renewing spermatogonia were
harmed (Brilhantaeet al., 2011). In few
Seminiferous tubules syncytitia/giant cell
formation was noticed. Similar findings were
also reported by Brilhantae et al., (2011).
Almost all the seminiferous tubules showed
severe atrophy as they are devoid of
epithelium. In Group 4 the testicular sections
on day 15 and 29 revealed active
spermatogenesis with several mitotic figures
in spermatogenic cells. In few seminiferous
tubules mild degenerative changes and
interstitial edema were noticed. Similar
findings were reported by Bashandy and
Amin (2012). Thus quercetin showed
cytoprotection due to its effect on prevention
of free radical production in testicular cells,
 Int.J.Curr.Microbiol.App.Sci (2017) 6(7): 2295-2306
so mechanical disruption of epithelium was
less. These findings were supported by
previous studies demonstrating that active
compounds present in quercetin has protective
effects against testicular damage caused by
different toxic agents such as carbon
tetrachloride (Sonmez et al., 2014), docetaxel
(Altintas et al., 2015), TCDD (Ciftci et al.,
2012) and FNT (Saber et al., 2015).
Ultra structurally Group 2 testes showed
prominent sub cellular changes like thinning
of basement membrane, distorted
spermatogenic cells, peripheral margination
of chromatin in spermatocytes, swollen
mitochondria, vacuolation of cellular
cytoplasm and presence of electron dense
lipid bodies during 15th day of experiment.
On 29th day severity was more with
additional features like indistinct cellular
junctions. These findings were in accordance
with Prahalathan et al., (2006).
These sub cellular changes could be due to
toxic action of doxorubicin on mitochondria,
nucleus and cell membranes as a part of
oxidative stress induced reaction which were
positively correlated with histological
parameters. Group 4 ultra-thin sections of
testes on 15th and 29th day revealed
regeneration and reconstruction of sub
cellular structures. There was increase in
spermatic cells of different shapes and sizes.
Interestingly on 15th day acrosomal cap
formation was evident which is indicative of a
positive action of ameliorative agent. Besides
this the other sub cellular changes like
vacuolar changes in cytoplasm were present
in most of the sections which are of lesser
extent.
The present study indicates reproductive
toxicity induced by DOX is related to
increased oxidative stress and quercetin a
potential antioxidant protects DOX induced
testicular toxicity.
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How to cite this article:

Divya, S., D. Madhuri, M. Lakshman and Gopal Reddy, A. 2017. Pathological and Ultra-
structural Changes in Testis of Rats due to Doxorubicin Toxicity and its Amelioration with
Quercetin. Int.J.Curr.Microbiol.App.Sci. 6(7): 2295-2306.
doi: https://doi.org/10.20546/ijcmas.2017.607.330

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